An Untargeted Metabolomics Approach to Study the Variation between Wild and Cultivated Soybeans.

The differential metabolite profiles of four wild and ten cultivated soybeans genotypes were explored using an untargeted metabolomics approach. Ground soybean seed samples were extracted with methanol and water, and metabolic features were obtained using ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS) in both positive and negative ion modes. The UHPLC-HRMS analysis of the two different extracts resulted in the putative identification of 98 metabolites belonging to several classes of phytochemicals, including isoflavones, organic acids, lipids, sugars, amino acids, saponins, and other compounds. The metabolic profile was significantly impacted by the polarity of the extraction solvent. Multivariate analysis showed a clear difference between wild and cultivated soybean cultivars. Unsupervised and supervised learning algorithms were applied to mine the generated data and to pinpoint metabolites differentiating wild and cultivated soybeans. The key identified metabolites differentiating wild and cultivated soybeans were isoflavonoids, free amino acids, and fatty acids. Catechin analogs, cynaroside, hydroxylated unsaturated fatty acid derivatives, amino acid, and uridine diphosphate-N-acetylglucosamine were upregulated in the methanol extract of wild soybeans. In contrast, isoflavonoids and other minor compounds were downregulated in the same soybean extract. This metabolic information will benefit breeders and biotechnology professionals to develop value-added soybeans with improved quality traits.

Quantitative proteomic analyses reveal the dynamics of protein and amino acid accumulation during soybean seed development.

Using high throughput tandem mass tag (TMT) based tagging technique, we identified 4172 proteins in three developmental stages: early, mid, and late seed filling. We mapped the identified proteins to metabolic pathways associated with seed filling. The elevated abundance of several kinases was observed from the early to mid-stages of seed filling, indicating that protein phosphorylation was a significant event during this period. The early to late seed filling stages were characterized by an increased abundance of proteins associated with the cell wall, oil, and vacuolar-related processes. Among the seed storage proteins, 7S (ß-subunit) and 11S (Gy3, Gy4, Gy5) steadily increased in abundance during early to late stages of seed filling, whereas 2S albumin exhibited a decrease in abundance during the same period. An increased abundance of proteases, senescence-associated proteins, and oil synthesis proteins was observed from the mid to late seed filling stages. The mid to late stages of seed filling was also characterized by a lower abundance of transferases, transporters, Kunitz family trypsin, and protease inhibitors. Two enzymes associated with methionine synthesis exhibited lower abundance from early to late stages. This study unveiled several essential enzymes/proteins related to amino acid and protein synthesis and their accumulation during seed development. All data can be accessed through this link: https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=38784ecbd0854bb3801afc0d89056f84. (Accession MSV000087577).

Proteomic Profiling of Fast Neutron-Induced Soybean Mutant Unveiled Pathways Associated with Increased Seed Protein Content.

Mutagenesis through fast neutron (FN) radiation of soybean resulted in a mutant with a 15% increase in seed protein content. A comparative genomic hybridization analysis confirmed that the mutant is lacking 24 genes located at chromosomes 5 and 10. A tandem mass tag-based proteomic profiling of the wild type and the FN mutant revealed 3,502 proteins, of which 206 proteins exhibited increased abundance and 214 proteins showed decreased abundance. Among the abundant proteins, basic 7S globulin increased fourfold, followed by vacuolar-sorting receptor and protein transporters. The differentially expressed proteins were mapped on the global metabolic pathways. It was observed that there was an enrichment of 29 ribosomal proteins, 16 endoplasmic reticular proteins, and several proteins in export metabolic pathways. The deletion of the sequence-specific DNA binding transcription factor along with 23 other genes may have altered the negative regulation of protein syntheses processes, resulting in an increase in the overall protein content of the mutant seed. This mutant is a valuable resource for researchers to understand the metabolic pathways that may affect an increase in seed protein content (the mass spectrometry data files were submitted to massive.ucsd.edu # MassIVE MSV000084228).

Enhanced separation and analysis of low abundant soy proteins by dual washing extraction process.

Soybean seeds provide a rich source of proteins, fats, carbohydrates, and micronutrients. Extraction and analysis of low abundant soybean seed proteins are challenging because of its complex seed composition. For characterizing various proteins, it is paramount to remove the other interfering components, primarily oils, and carbohydrates. In the present study, we used a sequential dual washing process initially with hexane to remove oil and non-polar interferences, followed by 80% ethanol washing to remove about 60% of the total soluble sugars. The extracted soluble sugars were quantified using a newly developed and validated high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD). This newly developed combined washings process significantly enhanced the separation of both low molecular weight and low abundant proteins using 1D (one dimensional)- and 2D (two dimensional) gel electrophoresis. The separated proteins were trypsinized and analyzed by using Bruker amazon speed ion trap mass spectrometer equipped with an ESI source. This combined washing process allowed the identification of 18 additional low abundant soy proteins as compared to the simple hexane washed samples. This purification process will allow researchers to identify and investigate the role of low molecular weight and low abundant proteins as it relates to plant functions, nutrition, and health.

Quantitative Proteomic Analysis of Low Linolenic Acid Transgenic Soybean Reveals Perturbations of Fatty Acid Metabolic Pathways.

To understand the effect of fatty acid desaturase gene (GmFAD3) silencing on perturbation of fatty acid (FA) metabolic pathways, the changes are compared in protein profiling in control and low linolenic acid transgenic soybeans using tandem mass tag based mass spectrometry. Protein profiling of the transgenic line unveiled changes in several key enzymes of FA metabolism. This includes enzymes of lower abundance; fabH, fabF, and thioestrase associated with FA initiation, elongation, and desaturation processes and LOX1_5, ACOX, ACAA1, MFP2 associated with ß-oxidation of α-linolenic acids pathways. In addition, the GmFAD3 silencing results in a significant reduction in one of the major allergens, Gly m 4 (C6T3L5). These results are important for exploring how plants adjust in their biological processes when certain changes are induced in the genetic makeup. A complete understanding of these processes will aid researchers to alter genes for developing value-added soybeans.

Genomic changes and biochemical alterations of seed protein and oil content in a subset of fast neutron induced soybean mutants.

BACKGROUND: Soybean is subjected to genetic manipulation by breeding, mutation, and transgenic approaches to produce value-added quality traits. Among those genetic approaches, mutagenesis through fast neutrons radiation is intriguing because it yields a variety of mutations, including single/multiple gene deletions and/or duplications. Characterizing the seed composition of the fast neutron mutants and its relationship with gene mutation is useful towards understanding oil and protein traits in soybean. RESULTS: From a large population of fast neutron mutagenized plants, we selected ten mutants based on a screening of total oil and protein content using near infra-red spectroscopy. These ten mutants were regrown, and the seeds were analyzed for oil by GC-MS, protein profiling by SDS-PAGE and gene mapping by comparative genomic hybridization. The mutant 2R29C14Cladecr233cMN15 (nicknamed in this study as L10) showed higher protein and lower oil content compared to the wild type, followed by three other lines (nicknamed in this study as L03, L05, and L06). We characterized the fatty acid methyl esters profile of the trans-esterified oil and found the presence of five major fatty acids (palmitic, stearic, oleic, linoleic, and linolenic acids) at varying proportions among the mutants. Protein profile using SDS-PAGE of the ten mutants did exhibit discernable variation between storage (glycinin and ß-conglycinin) and anti-nutritional factor (trypsin inhibitor) proteins. In addition, we physically mapped the position of the gene deletions or duplications in each mutant using comparative genomic hybridization. CONCLUSION: Characterization of oil and protein profile in soybean fast neutron mutants will assist scientist and breeders to develop new value-added soybeans with improved protein and oil quality traits.

Biochemical and Anatomical Investigation of Sesbania herbacea (Mill.) McVaugh Nodules Grown under Flooded and Non-Flooded Conditions.

Sesbania herbacea, a native North American fast-growing legume, thrives in wet and waterlogged conditions. This legume enters into symbiotic association with rhizobia, resulting in the formation of nitrogen-fixing nodules on the roots. A flooding-induced anaerobic environment imposes a challenge for the survival of rhizobia and negatively impacts nodulation. Very little information is available on how S. herbacea is able to thrive and efficiently fix N2 in flooded conditions. In this study, we found that Sesbania plants grown under flooded conditions were significantly taller, produced more biomass, and formed more nodules when compared to plants grown on dry land. Transmission electron microscopy of Sesbania nodules revealed bacteroids from flooded nodules contained prominent polyhydroxybutyrate crystals, which were absent in non-flooded nodules. Gas and ion chromatography mass spectrometry analysis of nodule metabolites revealed a marked decrease in asparagine and an increase in the levels of gamma aminobutyric acid in flooded nodules. 2-D gel electrophoresis of nodule bacteroid proteins revealed flooding-induced changes in their protein profiles. Several of the bacteroid proteins that were prominent in flooded nodules were identified by mass spectrometry to be members of the ABC transporter family. The activities of several key enzymes involved in nitrogen metabolism was altered in Sesbania flooded nodules. Aspartate aminotransferase (AspAT), an enzyme with a vital role in the assimilation of reduced nitrogen, was dramatically elevated in flooded nodules. The results of our study highlight the potential of S. herbacea as a green manure and sheds light on the morphological, structural, and biochemical adaptations that enable S. herbacea to thrive and efficiently fix N2 in flooded conditions.

Recent update on methodologies for extraction and analysis of soybean seed proteins.

Soybean is one of the best sources of plant protein. Development of improved soybean cultivars through classical breeding and new biotech approaches is important to meet the growing global demand for soybeans. There is a critical need to investigate changes in protein content and profiles to ensure the safety and nutritional quality of new soybean varieties and their food products. A proteomics study begins with an optimal combination of extraction, separation and detection approaches. This review attempts to provide a summary of current updates in the methodologies used for extraction, separation and detection of protein from soybean, the basic foundations for good proteomic research. This information can be effectively used to investigate modifications in protein content and profiles in new varieties of soybeans and other crops. © 2018 Society of Chemical Industry.

Identification of a Halotolerant Mutant via In Vitro Mutagenesis in the Cyanobacterium Fremyella diplosiphon.

Energy metabolism and photosynthetic pigment accumulation are affected by salt stress in cyanobacteria leading to cessation of growth. In this study, the effect of salinity on the freshwater cyanobacterium, Fremyella diplosiphon, was investigated and mutagenesis-based efforts were undertaken to enhance salt tolerance. Salinity at a concentration of 10 g/L sodium chloride (NaCl) inhibited growth of wild type F. diplosiphon under white, red, and green light. Efforts to enhance halotolerance resulted in a mutant that could survive in 20 g/L NaCl for 15 generations with no significant reduction in phycobiliproteins (phycocyanin, phycoerythrin, and allophycocyanin) or chlorophyll a. Gene expression measured by quantitative reverse transcription-polymerase chain reaction revealed a three-fold increase in tripartite ATP-independent periplasmic transporters (TRAP) solute receptor transcript in the mutant compared to wild type. Our discovery of a TRAP transporter system in F. diplosiphon and its possible role in salinity response enables growth in brackish waters, which enhances its potential for biotechnological applications.

Characterization of developmental- and stress-mediated expression of cinnamoyl-CoA reductase in kenaf (Hibiscus cannabinus L.).

Cinnamoyl-CoA reductase (CCR) is an important enzyme for lignin biosynthesis as it catalyzes the first specific committed step in monolignol biosynthesis. We have cloned a full length coding sequence of CCR from kenaf (Hibiscus cannabinus L.), which contains a 1,020-bp open reading frame (ORF), encoding 339 amino acids of 37.37 kDa, with an isoelectric point (pI) of 6.27 (JX524276, HcCCR2). BLAST result found that it has high homology with other plant CCR orthologs. Multiple alignment with other plant CCR sequences showed that it contains two highly conserved motifs: NAD(P) binding domain (VTGAGGFIASWMVKLLLEKGY) at N-terminal and probable catalytic domain (NWYCYGK). According to phylogenetic analysis, it was closely related to CCR sequences of Gossypium hirsutum (ACQ59094) and Populus trichocarpa (CAC07424). HcCCR2 showed ubiquitous expression in various kenaf tissues and the highest expression was detected in mature flower. HcCCR2 was expressed differentially in response to various stresses, and the highest expression was observed by drought and NaCl treatments.

Molecular characterization of ferulate 5-hydroxylase gene from kenaf (Hibiscus cannabinus L.).

The purpose of this study is to clone and characterize the expression pattern of a F5H gene encoding ferulate 5-hydroxylase in the phenylpropanoid pathway from kenaf (Hibiscus cannabinus L.). Kenaf is a fast-growing dicotyledonous plant valued for its biomass. F5H, a cytochrome P450-dependent monooxygenase (CYP84), is a key enzyme for syringyl lignin biosynthesis. The full length of the F5H ortholog was cloned and characterized. The full-length F5H ortholog consists of a 1,557-bp open reading frame (ORF) encoding 518 amino acids (GenBank Accession number JX524278). The deduced amino acid sequence showed that kenaf F5H had the highest similarity (78%) with that of Populus trichocarpa. Transcriptional analysis of F5H ortholog was conducted using quantitative real-time PCR during the developmental stages of various tissues and in response to various abiotic stresses. The highest transcript level of the F5H ortholog was observed in immature flower tissues and in early stage (6 week-old) of stem tissues, with a certain level of expression in all tissues tested. The highest transcript level of F5H ortholog was observed at the late time points after treatments with NaCl (48 h), wounding (24 h), cold (24 h), abscisic acid (24 h), and methyl jasmonate (24 h).

Metabolic Profiling of a Fast Neutron Soybean Mutant Reveals an Increased Abundance of Isoflavones.

A total of 718 metabolites were identified in leaves and seeds of the soybean (Glycine max (L.) Merr., Fabaceae) fast neutron (FN) mutant 2012CM7F040p05ar154bMN15, which was previously shown to have 21 genes deleted and higher protein content in seeds as compared to wild-type. Among the identified metabolites, 164 were found only in seeds, 89 only in leaves, and 465 in both leaves and seeds. Metabolites that exhibited higher abundance in the mutant leaf than in the wild type include the flavonoids afromosin, biochanin A, dihydrodaidzein, and apigenin. Mutant leaves also exhibited a higher accumulation of glycitein-glucoside, dihydrokaempferol, and pipecolate. The seed-only metabolites that were found in higher abundance in the mutant compared to the wild type included 3-hydroxybenzoate, 3-aminoisobutyrate, coenzyme A, N-acetyl-ß-alanine, and 1-methylhistidine. Among several amino acids, the cysteine content increased in the mutant leaf and seed when compared to the wild type. We anticipate that the deletion of acetyl-CoA synthase created a negative feedback effect on carbon dynamics, resulting in increased amounts of cysteine and isoflavone-associated metabolites. Metabolic profiling provided new insight into the cascading effect of gene deletions that helps breeders to produce value-added nutritional seed traits.

Gene expression profiling of the plant pathogenic basidiomycetous fungus Rhizoctonia solani AG 4 reveals putative virulence factors.

Rhizoctonia solani is a ubiquitous basidiomycetous soilborne fungal pathogen causing damping-off of seedlings, aerial blights and postharvest diseases. To gain insight into the molecular mechanisms of pathogenesis a global approach based on analysis of expressed sequence tags (ESTs) was undertaken. To get broad gene-expression coverage, two normalized EST libraries were developed from mycelia grown under high nitrogen-induced virulent and low nitrogen/methylglucose-induced hypovirulent conditions. A pilot-scale assessment of gene diversity was made from the sequence analyses of the two libraries. A total of 2280 cDNA clones was sequenced that corresponded to 220 unique sequence sets or clusters (contigs) and 805 singlets, making up a total of 1025 unique genes identified from the two virulence-differentiated cDNA libraries. From the total sequences, 295 genes (38.7%) exhibited strong similarities with genes in public databases and were categorized into 11 functional groups. Approximately 61.3% of the R. solani ESTs have no apparent homologs in publicly available fungal genome databases and are considered unique genes. We have identified several cDNAs with potential roles in fungal pathogenicity, virulence, signal transduction, vegetative incompatibility and mating, drug resistance, lignin degradation, bioremediation and morphological differentiation. A codon-usage table has been formulated based on 14694 R. solani EST codons. Further analysis of ESTs might provide insights into virulence mechanisms of R. solani AG 4 as well as roles of these genes in development, saprophytic colonization and ecological adaptation of this important fungal plant pathogen.

Protein profiling of fast neutron soybean mutant seeds reveals differential accumulation of seed and iron storage proteins.

A fast neutron (FN) radiated mutant soybean (Glycine max (L.) Merr., Fabaceae) displaying large duplications exhibited an increase in total seed protein content. A tandem mass tag (TMT) based protein profiling of matured seeds resulted in the identification of 4338 proteins. Gene duplication resulted in a significant increase in several seed storage proteins and protease inhibitors. Among the storage proteins, basic 7 S globulin, glycinin G4, and beta-conglycinin showed higher abundance in matured FN mutant seeds in addition to protease inhibitors. A significantly higher abundance of L-ascorbate peroxidases, acid phosphatases, and iron storage proteins was also observed. A higher amount of albumin, sucrose synthase, iron storage, and ascorbate family proteins in the mutant seeds was observed at the mid-stage of seed filling. We anticipate that the duplicated genes might have a cascading effect on the genome constituents, thus, resulting in increased storage and iron-containing protein content in the mutant seeds.

Two-dimensional proteome reference maps for the soybean cyst nematode Heterodera glycines.

2-DE reference maps of Heterodera glycines were constructed. After in-gel digestion with trypsin, 803 spots representing 426 proteins were subsequently identified by LC-MS/MS. Proteins with annotated function were further categorized by Gene Ontology. The results showed that proteins involved in metabolic, developmental and biological regulation processes were the most abundant.

Protein and metabolite composition of xylem sap from field-grown soybeans (Glycine max).

The xylem, in addition to transporting water, nutrients and metabolites, is also involved in long-distance signaling in response to pathogens, symbionts and environmental stresses. Xylem sap has been shown to contain a number of proteins including metabolic enzymes, stress-related proteins, signal transduction proteins and putative transcription factors. Previous studies on xylem sap have mostly utilized plants grown in controlled environmental chambers. However, plants in the field are subjected to high light and to environmental stress that is not normally found in growth chambers. In this study, we have examined the protein and metabolite composition of xylem sap from field-grown cultivated soybean plants. One-dimensional gel electrophoresis of xylem sap from determinate, indeterminate, nodulating and non-nodulating soybean cultivars revealed similar protein profiles consisting of about 8-10 prominent polypeptides. Two-dimensional gel electrophoresis of soybean xylem sap resulted in the visualization of about 60 distinct protein spots. A total of 38 protein spots were identified using MALDI-TOF MS and LC-MS/MS. The most abundant proteins present in the xylem sap were identified as 31 and 28 kDa vegetative storage proteins. In addition, several proteins that are conserved among different plant species were also identified. Diurnal changes in the metabolite profile of xylem sap collected during a 24-h cycle revealed that asparagine and aspartate were the two predominant amino acids irrespective of the time collected. Pinitol (D-3-O-methyl-chiro-inositol) was the most abundant carbohydrate present. The possible roles of xylem sap proteins and metabolites as nutrient reserves for sink tissue and as an indicator of biotic stress are also discussed.

Distinct cell surface appendages produced by Sinorhizobium fredii USDA257 and S. fredii USDA191, cultivar-specific and nonspecific symbionts of soybean.

Sinorhizobium fredii USDA257 and S. fredii USDA191 are fast-growing rhizobia that form nitrogen-fixing nodules on soybean roots. In contrast to USDA191, USDA257 exhibits cultivar specificity and can form nodules only on primitive soybean cultivars. In response to flavonoids released from soybean roots, these two rhizobia secrete nodulation outer proteins (Nop) to the extracellular milieu through a type III secretion system. In spite of the fact that Nops are known to regulate legume nodulation in a host-specific manner, very little is known about the differences in the compositions of Nops and surface appendages elaborated by USDA191 and USDA257. In this study we compared the Nop profiles of USDA191 and USDA257 by one-dimensional (1D) and 2D gel electrophoresis and identified several of these proteins by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and liquid chromatography-tandem MS (LC-MS/MS). Examination of the surface appendages elaborated by these two strains of soybean symbionts by transmission electron microscopy revealed distinct differences in their morphologies. Even though the flagella produced by USDA191 and USDA257 were similar in their morphologies, they differed in their flagellin composition. USDA257 pili resembled long thin filaments, while USDA191 pili were short, rod shaped, and much thinner than the flagella. 2D gel electrophoresis of pilus-like appendages of USDA191 and USDA257 followed by mass spectrometry resulted in the identification of several of the Nops along with some proteins previously undetected in these strains. Some of the newly identified proteins show homology to putative zinc protease and a LabA-like protein from Bradyrhizobium sp. ORS278, fimbrial type 4 assembly proteins from Ralstonia solanacearum, and the type III effector Hrp-dependent protein from Rhizobium leguminosarum bv. trifolii.

Natural variability in abundance of prevalent soybean proteins.

Soybean is an inexpensive source of protein for humans and animals. Genetic modifications (GMO) to soybean have become inevitable on two fronts, both quality and yield will need to improve to meet increasing global demand. To ensure the safety of the crop for consumers it is important to determine the natural variation in seed protein constituents as well as any unintended changes that may occur in the GMO as a result of genetic modification. Understanding the natural variation of seed proteins in wild and cultivated soybeans that have been used in conventional soybean breeding programs is critical for determining unintended protein expression in GMO soybeans. In recent years, proteomic technologies have been used as an effective analytical tool for examining modifications of protein profiles. We have standardized and applied these technologies to determine and quantify the spectrum of proteins present in soybean seed. We used two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS), and liquid chromatography mass spectrometry (LC-MS) for the separation, quantification, and identification of different classes of soybean seed proteins. We have observed significant variations in different classes of proteins, including storage, allergen and anti-nutritional protein profiles, between non-GMO cultivated and wild soybean varieties. This information is useful for scientists and regulatory agencies to determine whether the unintended expression of proteins found in transgenic soybean is within the range of natural variation.

A rapid and simple procedure for the depletion of abundant storage proteins from legume seeds to advance proteome analysis: a case study using Glycine max.

2-D analysis of plant proteomes containing thousands of proteins has limited dynamic resolution because only abundant proteins can be detected. Proteomic assessment of the non-abundant proteins within seeds is difficult when 60-80% is storage proteins. Resolution can be improved through sample fractionation using separation techniques based upon different physiological or biochemical principles. We have developed a fast and simple fractionation technique using 10 mM Ca(2+) to precipitate soybean (Glycine max) seed storage globulins, glycinin and beta-conglycinin. This method removes 87+/-4% of the highly abundant seed proteins from the extract, allowing for 541 previously inconspicuous proteins present in soybean seed to be more detectable (volume increase of >or=50%) using fluorescent detection. Of those 541 enhanced spots, 197 increased more than 2.5-fold when visualized with Coomassie. The majority of those spots were isolated and identified using peptide mass fingerprinting. Fractionation also provided detection of 63 new phosphorylated protein spots and enhanced the visibility of 15 phosphorylated protein spots, using 2-D electrophoretic separation and an in-gel phosphoprotein stain. Application of this methodology toward other legumes, such as peanut, bean, pea, alfalfa and others, also containing high amounts of storage proteins, was examined, and is reported here.

An efficient extraction method to enhance analysis of low abundant proteins from soybean seed.

Large amounts of the major storage proteins, beta-conglycinin and glycinin, in soybean (Glycine max) seeds hinder the isolation and characterization of less abundant seed proteins. We investigated whether isopropanol extraction could facilitate resolution of the low abundant proteins, different from the main storage protein fractions, in one-dimensional polyacrylamide gel electrophoresis (1D-PAGE) and two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). 1D-PAGE of proteins extracted by different concentrations (10%, 20%, 30%, 40%, 50%, 60%, 70% and 80%) of isopropanol showed that greater than 30% isopropanol was suitable for preferential enrichment of low abundant proteins. Analysis of 2D-PAGE showed that proteins which were less abundant or absent by the conventional extraction procedure were clearly seen in the 40% isopropanol extracts. Increasing isopropanol concentration above 40% resulted in a decrease in the number of less abundant protein spots. We have identified a total of 107 protein spots using matrix-assisted laser desorption/ionization time of flight mass spectrophotometry (MALDI-TOF-MS) and liquid chromatography-mass spectrometry (LC-MS/MS). Our results suggest that extraction of soybean seed powder with 40% isopropanol enriches lower abundance proteins and is a suitable method for 2D-PAGE separation and identification. This methodology could potentially allow the extraction and characterization of low abundant proteins of other legume seeds containing highly abundant storage proteins.